Method and apparatus to control the heterogeneous flow of bone cement and improve osseointegration of cemented implant

ABSTRACT

The present invention provides processes for combined applications of making grooves on an implant surface, applying MgO nanoparticles with PMMA cement, restricting the cement movement by PCL nanofiber and tethering biomolecules with PCL nanofiber to enhance mechanical stability and osseointegration of PMMA cement with bone. This is achieved through enhanced osteoconductive properties, roughness, and less viable fracture originating sites at the bone-cement interface. Such combined applications of nanoparticle and nanofiber on the mechanical stability and osseointegration of cemented implant is heretofore unknown, but as provided by the present invention can solve the debonding problem of cemented implant from bone.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims benefit of U.S. patent application Ser. No.15/674,309, filed Aug. 10, 2017 by the University of Central Oklahoma(Applicant,) entitled “Method and apparatus to control the heterogeneousflow of bone cement and improve osseonintegration of cemented implant”the entire disclosure of which is incorporated herein by reference inits entirety for all purposes. This application claims the benefit ofU.S. Provisional Patent Application No. 62/373,786 filed on Aug. 11,2016 in the name of Morshed Khandaker and Shahram Riahinezhad, which isexpressly incorporated herein by reference in its entirety for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under 8P20GM103447awarded by the United States National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to the field of prosthetics.More specifically, the invention relates to attachment of orthopedicimplants to bone using orthopedic bone cement. Cemented fixation oftitanium (Ti) with bone are commonly used in orthopedic surgeries.Cemented fixation, mainly used for osteoporotic bone, requires bonecement to hold the Ti based prostheses in place. Since implant looseningusually occurs in cemented joint replacement surgeries from lack ofosseointegration of implant (Ti or bone cement) with bone, clinicallybetter osseointegration of implant with bone is required to prevent orat least diminish loosening over time to preclude repeat surgeries. Thepresent invention solves the loosening problem of cemented implantsurgeries both orthopedic and orthodontic applications.

BACKGROUND OF THE INVENTION

Cemented fixation of an implant, mainly used for osteoporotic bone,requires bone cement to hold the implant in place. Although considerableadvances have already been made to improve the biological performance ofcement, the ideal long-term mechanical stability of a cemented implantis still not achieved. An ideal cementing material for cementedsurgeries should have surface energy and mechanical interlock to ensurea long-lasting fixation between implant-cement and cement-boneinterfaces. The critical task for creating a long lasting tissue-implantinterface resides in achieving the functional integration to mimicnative tissue-tissue failure response. Appropriate mechanical interlockand adequate osseointegration is present between the joining tissues atnatural tissue-tissue interfaces. Since bone cement is a bio-inertmaterial, in case of natural tissue-cement interface in cemented joint,the joining of cement with bone is done by mechanical interlock. Thegoal of this innovation is to increase the osseointegration at thetissue-cement interface by improving the bioactivity of cement so thatit will mimic native tissue-tissue failure response under functionalloading.

The debonding of the PolyMethylMethAcrylate (PMMA) cement from bone incemented joint replacement is frequently reported in literature. In thecase of total cemented joint replacements, implant loosening occurs dueto debonding of the bone-cement interface due to poor osseointegrationof bone cement with bone or weakening of bone due to local high stressedarea. Heterogeneous flow of bone cement around the implants due to theporosity of trabecular bone has been observed. Localized fractures mayoccur at the narrow confined tissue-cement interface by a relativelysmaller force in compare to failure force of bone due to thisheterogeneous flow of cement (FIG. 1). PMMA is a bio-inert material.Current trend of biomaterial research is focused on the addition ofbio-additives with cement to solve the debonding problem by improvingthe osseointegration of cements with bone. The purpose of thisinnovation is to coat PMMA at the bone/cement interface by nanofiberimmobilized with drugs to improve the biocompatibility PMMA cementwithout the diminishing the mechanical properties of PMMA at in vivocondition.

Nanofibers are a simple, scalable, inexpensive and supplementary surfacetreatment technique for biomaterials that have been implemented byvarious researchers. Most of research of the nanofiber applications oncement is focused on improving the mechanical properties of cementrather than improving the bioactivity of bone cement. For example,Wagner and Cohn used high performance polyethylene fibers as areinforcing phase in PMMA bone cement. The authors found that thesurface coating treatments of the Spectra 900 polyethylene fibersapparently did not significantly affect the mechanical properties of thePMMA bone cement. Saha and Pal found that addition of 1-2% by weight ofgraphite and up to 6% aramid fibers into PMMA cement reinforcedsignificantly the mechanical strength of PMMA. However, the previousauthors did not conduct cell viability studies to evaluate the effect oftheir fiber treatments on the biocompatibility of PMMA. Nanofibers canbe biomineralised by immobilization of functional proteins and mineralswith the fiber. Wu. et al. produced aligned poly(l-lactide)/poly(methylmethacrylate) binary blend fibers and mats loaded with a chimeric greenfluorescence protein having a bioactive peptide with hydroxyapatitebinding and mineralization property by pressurized gyration. Theprevious authors' research showed that nanofiber can have controllableinherent mineralization abilities through integrated bioactivity.However, no method has been proposed by which to apply nanofibermembrane on cement to improve its biomechanical properties. In ourresearch, we showed a technique to put the electrospun fiber membrane onthe surface of set PMMA cement. Our recent research manuscript inNanomaterial Journal titled “Use of Polycaprolactone ElectrospunNanofibers as a Coating for Poly (methylmethacrylate) Bone Cement”showed how to apply fiber membrane on cement for biomechanicalcharacterization (FIG. 2). But the problem is how a clinician is goingto apply such fiber membrane in real life for cemented surgeries. Therationale for using bone cement is that it is injected in the doughphase of mechanical properties during the polymerisation process. It isused as “grout” so a space filler to give either an interference fitbetween the implant and the supporting bone or to fill defects such aswhen it is used for cemented joint surgeries. In our innovation, we havedeveloped and described a process by which the nanofiber membrane of anyspecific size or shape, with or without drugs can be placed on thesurface of set cement. Our invented technique can also control the flowof cement into bone cavities using the flexibility and strength ofelectrospun nanofiber membrane.

SUMMARY OF THE INVENTION

Electrospinning is a process by which fibers with micron- to nanometerdiameters can be deposited on a substrate from an electrostaticallydriven jet of polymer solution through a needle. These fibers have ahigh surface area-to-volume ratio, which can be used to produce anelectrospun nanofiber (ENF) membrane for biomedical applications.Polycaprolactone (PCL) nanofibers can be produced using anelectrospinning process that is biocompatible and nontoxic. Anelectrospun nanofiber membrane (ENFM) has been developed in ourinnovation that can not only improve the osseointegration of cement withadjoining tissue, but also can control the flow of cement intotrabecular bone cavities. We have demonstrated the improvement ofbiomechanical performance of poly methyl methacrylate (PMMA) cement andtissue-cement interface resulting from the addition of antibacterial andosteoconductive nanoparticles (e.g. MgO, silver, TiO₂, ZnO) with PMMA.Our in vitro studies using bone cells and in vivo using rabbit modelshows that the ENFM coating increased the biocompatibility of cementthat lead to better mechanical stability and osseointegration of cementwith tissue. Further improvement of biomechanical performance of polymethyl methacrylate (PMMA) cement and tissue-cement interface wasachieved with the addition of antibacterial and osteoconductivenanoparticles (e.g. MgO, silver, TiO2, ZnO) with PCL ENFM.

Combined applications of making groove or ion deposition on implantsurface (co-pending application Ser. No. 15/467,652 by the presentinventor), applying nanoparticles additives with PMMA cement,immobilization of osteoconductive nanomaterials with ENF andconstruction of nanofiber membrane with adequate stiffness to controlthe cement movement in to the bone has be found to enhance mechanicalstability and osseointegration of PMMA cement with bone. Our researchhas demonstrated this method through clinical application of the use ofnanofiber membrane for cemented implant surgeries in an animal model.Such combined applications of nanoparticle and nanofiber on themechanical stability and osseointegration of cemented implant isheretofore unknown, but as provided by the methods of the presentinvention can solve the debonding problem of cemented implant from bone.

One objective of the present invention is to use grooving on Ti producedby the nanofabrication technique (disclosed in co-pending applicationSer. No. 15/467,652 by the present inventor and incorporated herein byreference), nanoparticles additives with bone cement, and bone growthprotein/minerals with PCL nanofiber membrane to improve theosseointegration of cemented implant from bone.

In one major aspect, microgrooves are fabricated on an implant bycontrolled formation of titanium nitride (TiN) ridges and themicrogrooves are anchored in bone by nanoparticles additives (NPA) (e.g.MgO, Hydroxyapatite, chitosan, etc.) incorporated with bone cement toproduce higher biomechanical advantages compared to non-grooved Tiimplants and non-NPA cemented implants due to increased biologicalcompatibility of treated Ti and cement.

In another aspect, microgrooves are coupled with poly-ε-caprolactonenanofiber membrane (PCL NFM) to improve the biomechanical performancesof Ti to advance in-vivo tissue-to-implant osseointegration and producefaster healing times.

In another aspect, microgrooves are coupled with growth factors (e.g.collagen) immobilized-poly-ε-caprolactone nanofiber membrane (CG-PCLNFM) and nanoparticles additives (NPA) poly-ε-caprolactone nanofibermembrane (NPA-PCL NFM) coating are coupled with the biomechanicalfunctions of Ti implant to produce higher biomedical advantages comparedto PCL NFM due to the increased osteoinductive and antimicrobial natureof the coatings.

In another aspect, ENF membrane can be used to act as resource for celladhesion matrix protein (e.g. fibronectin, cellulous) to the adjoiningbone tissue to produce better osseointegration with the cement surface.

In another aspect, prolonged antimicrobial and osteoinductive activitiesof PCL NFM is made possible by tethering the antimicrobial andosteoinductive molecules with PCL fiber (MgO, ZnO, Ag) in the ENF.

In another aspect, further improvement of cement-bone interface usingPCL ENF cup is made possible by immobilization of bone growth proteinand molecules (rhBMP, TGF-β) with the PCL ENF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a non-limiting diagram showing the heterogeneous flow of bonecement around the implants for a cemented implant surgery resulting fromthe porosity of bone: (A) histology image of a cemented implant and (B)a corresponding schematic representation. In the images the bold arrowsshow the existence of narrowly confined cement areas in the spongy bone.

FIG. 2 is a non-limiting diagram showing schematic representation ofmajor steps in the production methods for the present invention: step(a) single layer aligned PCL ENF membrane production, step (b)preparation of fiber coated cement test samples for surface andmechanical characterization, and step (c) preparation of fiber coatedcement test samples for cytocompatibility test samples.

FIG. 3 is a non-limiting diagram showing a schematic view of the mainsteps required for the application of ENF membranes in a cementedimplant surgery.

FIG. 4 is a non-limiting diagram showing a schematic view of the detailsteps required for the application of ENF membranes in a cementedimplant surgery.

FIG. 5 is a non-limiting diagram showing a schematic view of the methodof placing PCL ENF fiber cup in bone.

FIG. 6 is a non-limiting diagram showing a schematic view of the processfor the production of PCL ENF fiber cup for the fiber coated cementsurgery.

FIG. 7 is a non-limiting diagram showing a schematic view of the processfor the production of PCL ENF fiber membrane with collagen and/orfibronectin.

FIG. 8 is a non-limiting diagram showing a schematic view of theproduction of PCL ENF fiber cup with MgO nanoparticle tethered PCL.

FIG. 9 is a captured image showing (A) the electrospin setup to producePCL ENF membrane production, (B) membrane spun on to a custom made rod,and (C) the produced cylindrical cup shape membrane extracted from theperimeter of the rod.

FIG. 10 is a captured image showing (A) the holder used for the carrierof fiber cup during animal surgery, (B) the cylindrical shape needleused inject cement after placing the fiber cup in the hole created forcementing an implant, (C) syringe used to push the cement in to the cupinside the surgery hole.

FIG. 11 is a captured image showing the fixation of a in vivo sample ina mechanical tester for the pull out tension test: (A) potting of thetitanium anchored by PMMA bone cement sample in the mechanical testholder and (B) an implanted titanium anchored by PMMA bone cement usingPCL ENF fiber cup after the mechanical test.

FIG. 12 is a non-limiting diagram showing the results of in vitro cellviability experiments on control and PCL ENF coated PMMA samples. (A)Mean cell adhesion density (±standard error) and the percentage of cellproliferation (±standard error) for the control and ENF coated PMMAgroups after 48 h of cell culture. Data are presented with n=14 for bothsamples. (B) Mean amount of mineralization (±standard error) and meanamount of osteonectin (±standard error) for the control and ENF coatedPMMA groups. Note: * p<0.05 (compared to control).

FIG. 13 is a non-limiting diagram showing results of pull out tensiontest on three group of samples: (A) control, (B) ENF membrane, and (C)MgO NPA incorporated Ti/cement samples.

FIG. 14 is a non-limiting image showing results of histologicalexperiments. (A) Control Ti-cement samples, and (B) ENF membrane coveredTi-cement samples

FIG. 15 is a non-limiting diagram showing results of the tethering of 5wt % (A) MgO, (B) ZnO and (C) TiO₂ immobilized PCL ENF.

FIG. 16 is a non-limiting diagram showing results of the application ofthe innovation on the cemented hip surgery

DETAIL DESCRIPTION

The present invention provides a novel cementing technique to solve thedebonding problem and improve the mechanical stability andosseointegration of cemented implant with bone. Implants can be coatedwith a functional coating to increase the osteoinductive properties, andthereby to improve osseointegration of an implant. Methods (incorporatedherein by reference in their entirety) disclosed by the present inventorin U.S. Pat. No. 9,359,694 and co-pending U.S. application Ser. Nos.15/439,650 and 15/467,652 provide a set of steps (e.g. grooving, plasmaoxidizing) by which a nanofiber membrane, composed of Collagenglycosaminoglycan (CG) and Polycaprolecton (PCL) electrospun nanofiber(ENF) can be coated on Ti, a widely-used orthopedic and orthodonticimplant material. Both in vitro and in vivo evidence indicated thatmachine sawing of microgrooves on titanium (Ti) implants and coating themicrogrooves with collagen-poly-ε-caprolactone nanofiber matrix (CG-PCLNFM) significantly improves mechanical stability and osseointegration ofTi (M Khandaker, S. Riahinezhad, W. Williams, R. Wolf, “Microgroove andCollagen-poly(e-caprolactone) Nanofiber Mesh Coating Improves theMechanical Stability and Osseointegration of Titanium Implants.”Nanomaterials 2017, 7 (6), 145; doi: 10.3390/nano7060145). Asignificantly improved osseointegration of CG-PCL NFM coated Ti overnon-coated Ti was observed during the development of the disclosedmethods (Khandaker M, Riahinezhad S, Li Y, Sultana F, Morris T, VaughanM, Wolf R, Williams W. “Effect of collagen-polycaprolactoneextracellular matrix on the in vitro cytocompatibility and in vivo boneresponses of titanium.” Journal of Medical and Biological Engineering,38, 1-14, DOI 10.1007/s40846-017-0312-7, NIHMSID 895164).

Referring to FIG. 1, image A and corresponding diagram B shows aclinical problem where heterogeneous flow of bone cement around theimplant to the adjacent bone tissue has been observed due to theporosity of bone. Since significantly more cracks are associated withthe interdigitated area and the cement/bone interface than with theimplant/cement interface, there is a high probability that localizedfractures may occur at the narrowly confined cement/bone interfacesshown in corresponding diagram B in FIG. 1 due to this heterogeneousflow of cement. A method of reducing the localized fractures due to theheterogeneous flow of bone cement at the tissue-cement interface by afunctional nanofiber coating on cement has been developed in the presentinvention.

Referring to FIG. 2, a non-limiting diagram shows major steps in a novelprocess for fabrication of ENF fiber coating PMMA samples ofbiomechanical characterization: step (a) single layer aligned PCL ENFmembrane production, step (b) preparation of fiber coated cement testsamples for surface and mechanical characterization, and step (c)preparation of fiber coated cement samples for cytocompatibility testsamples. This process was developed to produce test samples forexperimental use in our clinical research directed to improving implantstrength and stability. The process at (a) shows fabrication of ENFfiber coating PMMA samples of biomechanical characterization. A glassslide 21 (25×75×1 mm) was coated with aligned PCL nanofibers using anelectrospinning setup 22. PCL pellets (7.69 wt %) were mixed withacetone in an ultrasonic mixer (Sonics & Materials, Inc., Newtown,Conn., USA). The sonication process was carried out at approximately 60°C. for 30 min. The solution was poured into a glass syringe 23 on aninfusion pump (Harvard Apparatus, mode # PHD ULTRA) for the PCL fiberproduction. The PCL solution was ejected from the glass syringe 23through an electrically-charged needle 24 (G blunt needle, 25 mm length,model # BX 25). The needle 24 was positively-charged by a high voltage(15 kV) DC power source 25 (Gamma High Voltage Research, Inc., model #ES 30 series) and two parallel wires were negatively-charged. Thealigned PCL fibers were collected between the two parallel wires. Tocollect multiple layers of aligned fiber, the top surface of the glassslides 21 touched the aligned fiber stream, moved up, and then movedforward to repeat the process to collect twenty four layers of fibers(˜1.6 micrograms) on the glass sides 21.

At process (b) in FIG. 2, a glass slide 26 without and with PCL ENF wassecured on the bottom of a mold 27 using double-sided tape to preparethe control and ENF coated cement samples, respectively. According tothe manufacturer recommendations, the PMMA solution was prepared by handmixing 2.2 g of PMMA powder with 1.1 mL of methyl methacrylate (MMA)monomer using a powder:monomer ratio of 2:1. All solutions were cured ina custom-made aluminum mold 27 to prepare a solid block of PMMA sampleof size 25×20×2 mm. Cement was poured into the chamber of the mold 27.Another glass side 26 was placed on top of the mold. Weights werestacked on the mold 27 to cure the cement under 60 kPa pressure(clinically applied pressure during orthopedic surgeries). The pressurewas initiated at three minutes after the onset of mixing (although itmay be possible to adjust the period) and was sustained throughout thecuring period (approximately 15 min).

Referring again to FIG. 2 at process (b), to prepare blocks of controland ENF coated PMMA samples for the mechanical tests, a 20×25×2 mmcontrol PMMA blocks was used. Mechanical test blocks were also used forthe surface topographical analysis using confocal microscopy. Since bothPCL ENF and PMMA cement have a white color, separate 20×25×2 mm ENFcoated PMMA blocks were prepared for SEM imaging and mechanical tests.To prepare an ENF coated PMMA sample for SEM imaging, the PMMA solutionwas mixed with a red-colored dye before being poured into the mold. Toprepare ASTM F417-78 standard flexural [29] test samples, (20×4×2) mmblocks were cut from the (20×25×2) mm block using a Buehler Isometlow-speed cutter. A (102×0.31×12.7) mm wafering blade was used forcutting the samples.

Referring to FIG. 2 at process (c), to prepare cytocompatibility of thecontrol and ENF coated PMMA samples a custom made well 28 were prepared.PCL ENF were collected between the wires in process (a) until a fibrouscloth appeared. A 10 mm diameter PCL fiber disc was cut from the clothusing a punch (FIG. 12a ). PMMA specimens were prepared by mixing 0.5 gof PMMA beads with 0.25 mL of MMA. All PMMA samples, while stillpliable, were divided into 4 parts by a knife and were poured in thewell. Each part of the samples was hand pressed during curing by aflat-ended 9.565 mm diameter highly polished round bar 29. The round bar29 has clearance fits on the wells of the well plate. To prepare the ENFcoated PMMA sample, a 10 mm diameter PCL fiber disc was placed on thecement and again pressed by the round bar 29 to attach the PCL fiber onthe top of the PMMA. The sample wells were kept sterile in a biologicalsafety cabinet under ultraviolet (UV) light for subsequent cell culture.

Referring now to FIG. 3, a non-limiting diagram of the method of thepresent invention is shown comprising five major steps: (A) Surgeryspecification; (B) Prepare Implant; (C) Prepare fiber membrane; (D)Prepare bone cement; and (E) Cementing of implant membrane.

Referring now to FIG. 4, the detail process in each step shown in FIG. 3is illustrated. For a cemented implant surgery (ex. screw implant), thespecification for surgery (e.g. hole dimension, implant size) must beknown—step (A). The surgery specification determines the size and shapeof ENF membrane that needs to be manufactured [see step (C)]. Thepresent invention implements a set of grooves/ridges on a Tiimplant—step (B) and fabricates a PCL ENF cylindrical membrane—step (C),that is placed in to the surgery hole (formed cavity) for the cementedimplant surgery. The grooves/ridges create mechanical interlock at theTi/cement interface and PCL ENF cylindrical membrane createsosteoinductive and osteoinductive microenvironment at the bone/cementinterface for the bone growth. Our experimentation demonstrated thatmechanical stability of Ti/cement samples having microgrooves on Ti(5.14±0.68 MPa, n=6) that were created by machine sawing weresignificantly higher (15 times) compared to Ti/cement samples withoutmicrogrooves on Ti (0.34±0.14 MPa, n=6) due to the increase of Ti-cementcontact area by microgrooving. Higher and more controlled Ti-bonecontact area can be created by plasma nitrogen deposition on Ti atselective regions which will create TiN ridges or other ion depositiontechnique.

The ENFM cylindrical membrane (e.g., ENFM cup) was inserted into thehole (formed cavity). PMMA cement was prepared by hand mixing PMMA andMMA monomer with and without MgO nanoparticles using bead: monomer ratioof 2:1—step (D). Referring to step (E), the method of inserting the ENFMcup into the hole (formed cavity) in the bone is indicated, where theENFM cup is placed over a rod and inserted into the hole. Cement isinjected into the ENFM cup, the implant is inserted and the cement iscured for approximately 5 minutes.

Referring to FIG. 5, the non-limiting diagram shows the process of thepresent invention where collagen and/or Fibronectin is added to the ENFMcup 51. The ENFM cup is placed into a hole (formed cavity) 54 using arod 52 sized properly for that purpose. Bone cement is added byinjecting it into the ENFM cup 51 using a syringe (see FIG. 10 C). Theimplant 53 is then inserted into the ENFM cup 51 positioned in the hole(formed cavity) 54 in the bone.

In our clinical studies, bilateral implantations were performed underanaesthetization on both legs of rabbits. A 2.96 mm diameter and 6 mmdeep hole 54 was made by a hand drill in the rabbit femur. The PCL ENFMcup 51 was inserted into the hole 54 of a rabbit femur at theepiphyso-metaphyseal junction. The cement in the dough phase ofmechanical properties during the polymerisation process was injectedinto the hole 54 of the ENFM cup 51 by a syringe (see FIG. 1 C).Subsequently, the implant 53 was hand-pressed into the cement.

Referring now to FIG. 6, the schematic of the process for the productionof PCL ENFM cup 61 is shown. Our developed electrospin unit 62 was usedto create a cylindrical PCL ENFM cup 61 (length 7 mm, inside diameter2.7 mm and thickness 0.1 mm) (FIG. 9 image C) by spraying PCL nanofiberson a rotating, round-shape collector 63 kept rotating by an electricmotor 64. ENFM cup 61 provides a membrane that can act as resource forcell adhesion matrix protein (collagen, fibronectin) and antimicrobialagents (MgO, ZnO, Ag) to the adjoining bone tissue to have betterosseointegration with the cement surface.

Referring to FIG. 7, a non-limiting diagram shows the process of thepresent invention for immobilization of cell adhesion matrix protein(collagen and/or fibronectin). A ENFM cup 71 sized to fit a specifichole (formed cavity) in bone is produced by electrospinning fibers 75onto a rapidly spinning cylindrical rod 72 with sufficient layers toproduce a stiffened cylinder as a formed ENFM cup 71. Collagen and/orfibronectin is added 73 to the ENFM 71 cup. FN is a multifunctionalprotein most abundantly found in the ECM under dynamic remodelingconditions such as bone healing and development. FN has a large bindingdomain for attaching growth factor proteins such as rhBMP, TGF-β. FNreinforces perimatrix formation, where it serves as a biological gluemediating interaction between cells and ECM proteins. FN can beimmobilized on Ti by tresyl chloride-activation process. FN contains aCG binding domain, so it can be polymerized into CG-PCL NFM.

In our clinical studies, cell viability tests were conducted on CG-PCLNFM coated samples with and without the plasma FN coating on Ti. Resultsshowed reduced amount of cell attachment (p>0.05), but significantimprovement of cell proliferation in NFM due to FN coating on Ti(p<0.05) suggesting that FN coating on Ti can further improve thebiological functions of our NFM. Cellular FN can be used instead ofplasma FN to increase the cell attachment on Ti, since cellular FN hasbetter functionality for the regulation of bone scaffolding protein andhigher adhesiveness than plasma FN.

Referring to FIG. 8, a non-limiting diagram illustrates the process ofimmobilization of nanoparticle additives (NPA) with PCL nanofiber. Apreferred method is shown as a schematic of the process for productionof PCL ENFM cup 81 using MgO nanoparticle tethered PCL. ENFM cup 81 isproduced by electrospinning fibers 82 onto a rapidly spinningcylindrical rod 83 with sufficient layers to produce a stiffenedcylinder. Using the methods of the present invention, we have tetheredMgO NPs with PCL nanofiber. MgO NPs were sonicated 84 for a period of 30minutes in acetone and then another 30 minutes with PCL beads to mix PCLwith the solution. The MgO NP-PCL solution was used to producenanofibers using an electrospun unit 85. In our research, we have usedMgO, ZnO, Ag nanoparticle additives, which have osteoconductive andantimicrobial properties. The above nanomaterials are widely used tofabricate efficient gas sensors for the detection of various hazardousand toxic gases. For example, MgO is used for SO₂ gas sensors [Lee etal, Sensors and Actuators B: Chemical, vol. 160, pp. 1328-1334, Dec. 15,2011], TiO₂ NP is used for low-temperature CO₂ gas sensors [Mardare etal, Ceramics International, vol. 42, pp. 7353-7359, May 1, 2016], ZnO NPis used for NO2 gas sensors [Kumar et al, Nano-Micro Letters, vol. 7,pp. 97-120, 2015//2015]. These NPAs can be tethered with the ultrafinefibers (see FIG. 15 images A, B, C) produced via electrospinning as athree-dimensional structured fibrous membrane with controllable porestructure and high specific surface area to produce membrane that can beused not only as a precision gas sensing device, but also as biomedicalmaterials such as suture, skin grafting, protective coating on implant.The PCL nanofibers with NPA (MgO, TiO2, ZnO) were prepared by dissolvingPCL and NPA (5% by wt of PCL) in 99.9% acetone (1000% by wt of PCL) andsonicating it for 30 min in 130 W and 20 kHz sonicator at 60% amplitude.Adding 5% of nano particles was discovered by trial and error methodbased on amount of MgO nano particles as it has the least density amongthe three.

Referring now to FIG. 9, a photograph of a PCL ENFM cup (image C)produced using the methods of the present invention is presented. ImageA shows the electrospinning setup, and image B shows the metal rod on towhich PCL nanofibers were collected to form the ENFM cup shown in imageC. The exemplary fabricated cup shown has a length of 7 mm, insidediameter of 2.6 mm and PCL ENF membrane thickness of 0.2 mm. The size ofthe cup produced corresponds to the dimensions of the hole (formedcavity) created in bone to receive a specific implant.

Referring now to FIG. 10, image (A) shows a custom made holderfabricated for our clinical studies and used to carry the ENFM cup understerilize condition. A sterile tweezer was used to deliver the ENFM cupcontaining a needle [see image (B)] to a veterinarian for implantationof an implant by manually created defect site in rabbit femur. A 10 mlsyringe shown in image C was loaded with bone cement. The needle withENFM cup was secured with syringe. A 2.96 mm diameter and 6 mm deep holewas made by a hand drill in rabbit femur. The ENFM cup was inserted intothe hole. PMMA cement was poured on the hole of the cup. Subsequently,the Ti wire was hand pressed into the cement.

Referring now to FIG. 11, an implanted titanium rod anchored by PMMAbone cement using PCL ENFM cup is shown before (image A) and after amechanical test (image B). It is clear from image B that our inventedPCL ENFM cup works for in vivo animal study and the cements werecontained by PCL ENFM cup.

Referring to FIG. 12, graph A compares adhesion proliferation for thetypes of cement samples used in testing. Graph B shows mineralizationand osteonectin adsorption for the types of cement samples used intesting. We have observed increased cytocompatibility properties(adhesion, proliferation, and protein adsorption) of the ENF coated PMMAimplants compared to PMMA. This is because higher cell functions werecreated via better cell signaling arising from the cell-cell contact andthe cell-ENF components in the ENF coated PMMA samples. Cell signalsdepend upon the physical (micro- or nano-structured surface topography,composition of ENF) and chemical properties of ENF. There existsdifferences of the physio-chemical properties between the control andENF coated PMMA samples. The PCL nanofibers on PMMA lead to differentphysical characteristics viz. porosity and density due to thedistribution of the PCL fiber. PCL in the ENF coated PMMA created alarger surface area that provided more cell binding sites. Additionally,PCL ENF can absorb numerous proteins or minerals akin to a cell membranereceptor, thus favoring cytocompatibility properties for the ENF coatedPMMA samples compared to the control.

Referring now to FIG. 13, in our clinical studies, three kinds ofTi/cement samples were prepared (A, B, C). They are Ti-PMMA (referred ascontrol—A), with ENFM cup Ti-PMMA cement (referred as ENF membrane—B),with (C) Ti-PMMA cement where cement is anchored to bone via PCL ENF andMgO nanoparticles mixed with PMMA cement (referred as MgO +ENFmembrane—C). For MgO +ENF membrane Ti/cement samples, PMMA cement wasprepared by mixing 10 wt % of MgO nanoparticles with PMMA cement.Mechanical and CT scan images were conducted on the samples using anestablished method. We have found that both PCL ENFM cup with andwithout MgO incorporated cement significantly improves the mechanicalstability of Ti/cement-bone joints. The ENF membrane acts as resourcefor bone growth molecules and antimicrobial agents to the adjoining bonetissue to have better osseointegration with the cement surface.

Referring to FIG. 14, a non-limiting image is presented showing resultsof histological experiments. (A) Control Ti-cement samples, and (B) ENFmembrane covered Ti-cement samples. The present invention has proventhrough experimentation to be successful in increasing the in vivomechanical stability (see FIG. 13) and no adverse effect on in vivoosseointegration as shown in the images of FIG. 14. Better MgOnanoparticles on in vivo mechanical stability is made possible bytethering MgO nanoparticles with PCL fiber using the methods of thepresent invention. All these observations and prediction are new and notreported in published literature or related art.

Referring to FIG. 15, nanoparticle drugs (osteoconductive andantibacterial) are shown (white dots) tethered with nanofiber. Using themethods of the present invention (FIG. 8), we have successfully tetheredMgO, ZnO and TiO₂ NPs with PCL nanofiber. The MgO NP-PCL, MgO NP-PCL andTiO2 NP-PCL solutions were used to produce MgO, ZnO and TiO2 tetheredPCL nanofibers using an electrospun unit. The attachment of the nanoparticles was confirmed by taking SEM images of the samples (FIG. 15images A, B, C). The sample was kept under the running water flow for aminute and images were taken again to confirm the nano particle'sattachment. It has been observed from animal studies that coating ofmicrogrooves on titanium with collagen (CG)-poly(ε-caprolactone) (PCL)nanofiber mesh (NFM) improves the mechanical stability andosseointegration of titanium (Ti). Moreover, histomorphometric analysisshowed that bone ingrowth to Ti surface increased by coatingmicrogrooves with the CG-PCL NFM. Further, NFM can be a carrier ofnanoparticle minerals at the implant sites that can influence themechanical stability and osseointegration of Ti with bone. We haveinvestigated whether mixing of MgO nanoparticle with CG-PCL NFM has anyinfluence on the mechanical stability and osseointegration of Ti. Wehave measured the effect of MgO nanoparticles on the in vitrocytocompatibility properties (osteoblast cell adhesion, proliferation,differentiation and protein adsorption) of CG-PCL NFM coated Ti. We havealso measured the effect of MgO nanoparticles on the in vivo mechanicalstability and osseointegration of CG-PCL NFM coated Ti with bone. Ourresearch found that cell adhesion/proliferation, andmineralization/protein adsorption of MgO NP added CG-PCL NFM coated Tiwas significantly higher compared to CG-PCL NFM (p<0.05). Both CG-PCLand CG-MgO-PCL treatment on Ti significantly influenced the in vivomechanical stability of the Ti as compared to groove only samples, butthere is no significance difference of mechanical stability foundbetween CG-PCL and CG-MgO-PCL treated samples. Mechanical results showedthat shear strength of Ti with bone for MgO added CG-PCL NFM coated(5.97±0.65 MPa, n=6) was higher compare to fracture strength of TiCG-PCL NFM (4.79±0.39 MPa, n=6) (p>0.05). Local delivery of protein andmineral to titanium is possible using the methods of the presentinvention via CG-PCL electronspun nanofiber matrix coating forcementless implant surgery. Similarly, local delivery of protein andmineral to cement with ENF fiber coating is possible using the methodsof the present invention to tether nanofibers with MgO, ZnO and TiO₂biomolecules (FIG. 15). Tethering nanofibers with MgO, ZnO and TiO₂biomolecules creates a better osteoconductive and antimicrobial platformat the cement/bone interface that reduces implant loosening byincreasing osseointegration and decreasing infection. The biologicalproperties of a functional coating can be further improved by addinggrowth factors, genes, and other biomolecules (such as hydroxpapatite,bisphosphonate) to create a truly osteoinductive platform at thecement/bone interface. Currently, no published research has been foundreporting adding the above factors to PCL ENF. Without the addition ofbone growth molecules, the invention shows significant improvement ofmechanical stability. Even further enhancement of mechanical stabilityand osseointegration is achieved by adding bone growth molecules orproteins with the PCL ENF in accordance with the methods of the presentinvention.

Referring to FIG. 16, the method provided by the present invention canbe used to apply ENF membrane to a cemented hip implant 161, where theshape and size of the ENF membrane 162 can be the same as the shape andsize of the hole drilled for the anchor of the cemented hip implant 161.A custom made aluminum block can be machined to the shape of the hole.ENF fibers can be deposited on the custom made block. The membrane canbe placed as marked in FIG. 16 and subsequently cement and implant canbe pressed in to the hole (formed cavity).

Our experimentation revealed that the in vitro biocompatilibity ofcement is improved by addition of PCL ENF membrane with PMMA (FIG. 12).Experimentation revealed that the in vivo mechanical stability ofbone/PMMA interface is improved by addition of PCL ENF membrane cup andmagnesium oxide (MgO) nanoparticles with PMMA (FIG. 13). Potentialapplications in surgery for the methods provided by the presentinvention include osteoporotic bone where cement is used to hold theimplant, but due to the void space of the bone and to osteoporoticdisease, cement penetrates some of narrow channeled void space. Whencemented, such void space has higher probability of fracture by appliedphysiological loading. By using the PCL ENFM cup produced by the methodsof the present invention, filling the narrow void space by ENF membranewith local drug, such failure of cement at the bone-cement interface canbe avoided. In addition, the PCL ENF fiber material can serve asreservoir by tethering nanoparticles (FIG. 15) for the local delivery ofnanomedicine for the orthopedic bone disease.

The controlled fabrication of microgrooves on Ti surfaces by plasmanitriding is significant, since such grooving can be applied to complexshape implant surfaces such as hip and dental implants, which isimpossible by machine sawing. Our invented NFM coating can serve as areservoir for controlled release of antimicrobial and growth factormolecules for reducing infection and promoting osteogenesis at thecement/bone interface. Providing osteogenesis pathways and the enhancedactivities induced by NFMs will greatly facilitate bone repair.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as examples of embodiments. Elements and materials maybe substituted for those illustrated and described herein, parts andprocesses may be reversed, and certain features of the invention may beutilized independently, all as would be apparent to one skilled in theart after having the benefit of this description of the invention.Changes may be made in the elements described herein without departingfrom the spirit and scope of the invention as described in the followingclaims.

The invention claimed is:
 1. An electrospun nanofiber (ENF) membrane,comprising: a fiber matrix exhibiting a tubular shape having an insidespan and an outside span, said matrix formed by electrospinningnanofibers on to an elongated substrate rotated around a longitudinalaxis during electrospinning to produce said tubular shape; multiplelayers of nanofibers, each of said layers consisting of generallyaligned fiber strands and said fiber strands comprising each layer arecollectively positioned at an oblique angle to the fiber strands in eachadjacent layer to form said matrix, said matrix comprising no more than24 layers of nanofibers; and nanoparticle additives (NPA) immobilizedwith poly-ε-caprolactone (PCL) nanofiber.
 2. The electrospun nanofiber(ENF) membrane of claim 1, further comprising growth factors immobilizedcollagen-poly-ε-caprolactone nanofiber matrix (CG-PCL NFM).
 3. Theelectrospun nanofiber (ENF) membrane of claim 1, further comprisingfibronectin (FN) and magnesium oxide nanoparticles (MgO NPs) immobilizedCG-PCL NFM.
 4. The electrospun nanofiber (ENF) membrane of claim 3,wherein said FN is immobilized using a tresyl chloride-activationprocess.
 5. The electrospun nanofiber (ENF) membrane of claim 1, furthercomprising immobilized cell adhesion matrix protein (collagen,fibronectin) and bone growth molecules (rhBMP, TGF-(β).
 6. Theelectrospun nanofiber (ENF) membrane of claim 1, further comprisingtethered antimicrobial and osteoinductive molecules (MgO, ZnO, Ag). 7.The electrospun nanofiber (ENF) membrane of claim 1, further comprisingusing MgO, ZnO and TiO₂ biomolecules with PCL ENF membrane to create aosteoinductive and antimicrobial platform.
 8. The electrospun nanofiber(ENF) membrane of claim 1, wherein said membrane is constructed with atleast one end layer of nanofibers to form a cup.
 9. The electrospunnanofiber (ENF) membrane of claim 1, wherein said membrane is adapted toprovide mechanical stability and osseointegration ofPolyMethylMethAcrylate (PMMA) cement with bone in surgeries that use ametallic implant.
 10. The electrospun nanofiber (ENF) membrane of claim9, where porosity of said ENF membrane is sized to prevent penetrationof said PMMA cement into void space within said bone tissue.
 11. Theelectrospun nanofiber (ENF) membrane of claim 10, further comprisingnanoparticle additives (NPA) MgO, ZnO, and Ag tethered with PCL in theENF by dissolving PCL and NPA (4% to 6% and preferably 5% by weight ofPCL) in acetone (900% to 1100% and preferably 1000% by weight of PCL),sonicating the mixture, and electrospinning the PCL with NPA.
 12. Theelectrospun nanofiber (ENF) membrane of claim 11, where said membrane isformed as a cup using said ENF, and exhibits adequate stiffness andporosity to control movement of said cement into said bone.
 13. Theelectrospun nanofiber (ENF) membrane of claim 12, wherein said nanofibermembrane is adapted to be inserted into a formed cavity in said bone,and receive said PMMA cement and said implant.